Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacturing of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products. Lactococcus species including Lactococcus lactis are among the most commonly used lactic acid bacteria in dairy starter cultures. However, several other lactic acid bacteria such as Leuconostoc species, Pediococcus species, Lactobacillus species and Streptococcus species are also commonly used in food starter cultures.
A significant role of lactic acid bacteria is to render the fermented products microbiologically stable and to improve the taste and palatability of these products. It is generally recognised that genes, the expression of which are important to ensure that the addition of lactic acid bacteria to a starting material results in the desired fermentation effect, are found naturally or can be inserted on extrachromosomal DNA vectors including plasmids.
However, DNA vectors may be unstable, resulting in their loss from the cells. Accordingly, it is of pertinent industrial interest to provide vectors which are stably maintained in lactic acid bacterial starter cultures.
Presently used methods of stably maintaining (stabilising) vectors in a host cell include insertion of relatively large DNA sequences such e.g. antibiotic or bacteriocin resistance genes into the cell. In the art, such genes are also referred to as selection markers. However, it is well-known that the insertion of large DNA sequences involves the risk that other sequences are deleted from the vector. Furthermore, the use of resistance genes for maintaining the plasmid in the host cell implies that antibiotics or bacteriocins must be present in the cultivation medium. This is undesirable in the manufacturing of food and feed products. In addition, it is undesirable that live bacteria comprising antibiotic resistance genes are present in food products as such genes may be transferable to the indigenous gastro-intestinal microflora.
Consequently, there have been reported several attempts to develop so-called food-grade cloning vectors. In the present context, the term “food-grade” indicates that the vector consists essentially of DNA of lactic acid bacterial origin.
WO 91/09131 discloses a vector essentially consisting of lactic acid bacterial DNA wherein a gene coding for the bacteriocin nisin is used as a selectable marker. However, the selection of such a vector still requires that a selective compound is added to the cultivation medium.
As an alternative approach, it has been suggested to use vectors carrying a gene coding for a gene product that suppresses nonsense mutations in lactic acid bacteria.
In the in vivo synthesis of proteins occurring in the ribosomes, mRNA is translated into polypeptide chains. However, the mRNA codons do not directly recognise the amino acids that they specify in the way that an enzyme recognises a substrate. Translation uses “adaptor” molecules that recognise both an amino acid and a triplet group of nucleotide bases (a codon). These adaptors consist of a set of small RNA molecules known as transfer RNAs (or tRNAs), each of which is only 70 to 90 nucleotides in length. Such tRNA molecules contain unpaired nucleotide residues comprising a CCA triplet at one end of the molecule and, in a central loop, a triplet of varying sequence forming the so-called anticodon that can base-pair to a complementary triplet in the mRNA molecule, while the CCA triplet at the free 3′ end of the molecule is attached covalently to a specific amino acid.
The three nucleotide triplets UAG (amber codon), UGA (opal codon) and UM (ochre codon) do not code for an amino acid. These signals termed stop codons or “nonsense” codons, are involved in polypeptide chain termination. During translation, two protein factors (R1 and R2) recognise these triplets and effect release of the polypeptide chain from the ribosome-mRNA-tRNA complex.
Occasionally a mutation occurs in a cell resulting in a nonsense codon appearing within a gene, causing premature chain termination and the production of a protein fragment. Such fragments rarely have enzymatic activity.
The effect of such a nonsense mutation can be reversed or suppressed by a second mutation in a gene coding for a tRNA which results in the synthesis of an altered tRNA molecule. Such an altered tRNA recognises a nonsense codon and inserts an amino acid at that point in the polypeptide chain. The mutated tRNA-encoding gene is termed a suppressor gene and the altered nonsense mutation-suppressing tRNA which it encodes is generally referred to as a nonsense or termination suppressor. Such termination suppressors may be derived by single, double or triple base substitutions in the anticodon region of the tRNA.
Most mutations in a tRNA-encoding gene leading to the formation of a nonsense suppressor are located in the anticodon triplet and alter it to CUA, UUA or UCA. Such suppressors may be referred to as amber, ochre and opal suppressors, respectively. Following the rules of nomenclature of Demerec et al., 1966 which was suggested for termination (nonsense) suppressors in E. coli the symbol “sup” and assigned capital letters as gene designations, e.g. supB, supC or supZ, are used herein also to designate suppressor genes in lactic acid bacteria.
In Dickely et al. 1995, Johansen et al. 1995 and WO 95/10621 are disclosed plasmids containing a gene coding for a tRNA that is a suppressor for a nonsense mutation where the suppressor gene will function as a selectable marker when the nonsense mutation in the host strain for the plasmid is one which, in the absence of a corresponding suppressor gene, will render the host strain incapable of growing in a particular environments, such as e.g. milk or other food or feed products. The genes coding for suppressor tRNA are small and can be inserted without causing deletions of desired genes. Also, homologous recombination will not occur between supD and the chromosomal tRNA genes due to the small size.
The construction of the vector pAK89.1 that comprises a supD suppressor is described in Dickely et al. 1995 and WO 95/10621. However, this cloning vector contains a gene coding for erythromycin resistance and thus is not a food-grade vector.
Dickely et al. 1995 and WO 95/10621 disclose food-grade vectors based on the Lactococcus lactis derived nonsense suppressor, supB, as a selection marker. However, these vectors, pFG1 and pFG1.1, cause growth inhibition when present in host cells. It has also been found that these particular vectors, when present in lactic acid bacterial strains, are unstable and that the acidification rate of the host cells in milk is reduced as compared to wildtype strains and therefore these vectors are not suitable in industrial processes.
Accordingly, the prior art is not aware of nonsense suppressor containing food-grade vectors that are stably maintained in lactic acid bacterial strains and which do not adversely affect the growth and metabolic activity of the host strains.
The present invention provides a food-grade vector, comprising as the selection marker a nonsense suppressor gene. It was surprisingly found, that the vector, when it is present in a lactic acid bacterial strain comprising a nonsense mutation suppressible by the suppressor, substantially does not cause growth inhibition and permits the strain to acidify milk at essentially the same rate as that of the same strain not containing the vector.